PARP inhibitors enhance replication stress and cause mitotic catastrophe in MYCN-dependent neuroblastoma.

High-risk and MYCN-amplified neuroblastomas are among the most aggressive pediatric tumors. Despite intense multimodality therapies, about 50% of these patients succumb to their disease, making the search for effective therapies an absolute priority. Due to the important functions of poly (ADP-ribose) polymerases, PARP inhibitors have entered the clinical settings for cancer treatment and are being exploited in a variety of preclinical studies and clinical trials. PARP inhibitors based combination schemes have also been tested in neuroblastoma preclinical models with encouraging results. However, the expression of PARP enzymes in human neuroblastoma and the biological consequences of their inhibition remained largely unexplored. Here, we show that high PARP1 and PARP2 expression is significantly associated with high-risk neuroblastoma cases and poor survival, highlighting its previously unrecognized prognostic value for human neuroblastoma. In vitro, PARP1 and 2 are abundant in MYCN amplified and MYCN-overexpressing cells. In this context, PARP inhibitors with high 'PARP trapping' potency, such as olaparib or talazoparib, yield DNA damage and cell death preceded by intense signs of replication stress. Notwithstanding the activation of a CHK1-CDC25A replication stress response, PARP-inhibited MYCN amplified and overexpressing cells fail to sustain a prolonged checkpoint and progress through mitosis in the presence of damaged DNA, eventually undergoing mitotic catastrophe. CHK1-targeted inhibition of the replication stress checkpoint exacerbated this phenotype. These data highlight a novel route for cell death induction by PARP inhibitors and support their introduction, together with CHK1 inhibitors, in therapeutic approaches for neuroblastomas with high MYC(N) activity.

Aurora-A and ch-TOG act in a common pathway in control of spindle pole integrity.

Mitotic spindle assembly is a highly regulated process, crucial to ensure the correct segregation of duplicated chromosomes in daughter cells and to avoid aneuploidy, a common feature of tumors. Among the most important spindle regulators is Aurora-A, a mitotic centrosomal kinase frequently overexpressed in tumors. Here, we investigated the role of Aurora-A in spindle pole organization in human cells. We show that RNA interference-mediated Aurora-A inactivation causes pericentriolar material fragmentation in prometaphase, yielding the formation of spindles with supernumerary poles. This fragmentation does not necessarily involve centrioles and requires microtubules (MTs). Aurora-A-depleted prometaphases mislocalize the MT-stabilizing protein colonic hepatic tumor-overexpressed gene (ch-TOG), which abnormally accumulates at spindle poles, as well as the mitotic centromere-associated kinesin (MCAK), the major functional antagonist of ch-TOG, which delocalizes from poles. ch-TOG is required for extrapole formation in prometaphases lacking Aurora-A, because co-depletion of Aurora-A and ch-TOG mitigates the fragmented pole phenotype. These results indicate a novel function of Aurora-A, the regulation of ch-TOG and MCAK localization, and highlight a common pathway involving the three factors in control of spindle pole integrity.

Ran induces spindle assembly by reversing the inhibitory effect of importin alpha on TPX2 activity.

The small GTPase Ran, bound to GTP, is required for the induction of spindle formation by chromosomes in M phase. High concentrations of Ran.GTP are proposed to surround M phase chromatin. We show that the action of Ran.GTP in spindle formation requires TPX2, a microtubule-associated protein previously known to target a motor protein, Xklp2, to microtubules. TPX2 is normally inactivated by binding to the nuclear import factor, importin alpha, and is displaced from importin alpha by the action of Ran.GTP. TPX2 is required for Ran.GTP and chromatin-induced microtubule assembly in M phase extracts and mediates spontaneous microtubule assembly when present in excess over free importin alpha. Thus, components of the nuclear transport machinery serve to regulate spindle formation in M phase.

Regulated Ran-binding protein 1 activity is required for organization and function of the mitotic spindle in mammalian cells in vivo.

Ran-binding protein (RanBP) 1 is a major regulator of the Ran GTPase and is encoded by a regulatory target gene of E2F factors. The Ran GTPase network controls several cellular processes, including nucleocytoplasmic transport and cell cycle progression, and has recently also been shown to regulate microtubule nucleation and spindle assembly in Xenopus oocyte extracts. Here we report that RanBP1 protein levels are cell cycle regulated in mammalian cells, increase from S phase to M phase, peak in metaphase, and abruptly decline in late telophase. Overexpression of RanBP1 throughout the cell cycle yields abnormal mitoses characterized by severe defects in spindle polarization. In addition, microinjection of anti-RanBP1 antibody in mitotic cells induces mitotic delay and abnormal nuclear division, reflecting an abnormal stabilization of the mitotic spindle. Thus, regulated RanBP1 activity is required for proper execution of mitosis in somatic cells.

Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation.

Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindle apparatus. During cell division, the nucleation of cytosolic microtubules is prevented and spindle microtubules nucleate from centrosomes (in mitotic animal cells) or around chromosomes (in plants and some meiotic cells). The molecular mechanism by which chromosomes induce local microtubule nucleation in the absence of centrosomes is unknown, but it can be studied by adding chromatin beads to Xenopus egg extracts. The beads nucleate microtubules that eventually reorganize into a bipolar spindle. RCC1, the guanine-nucleotide-exchange factor for the GTPase protein Ran, is a component of chromatin. Using the chromatin bead assay, we show here that the activity of chromosome-associated RCC1 protein is required for spindle formation. Ran itself, when in the GTP-bound state (Ran-GTP), induces microtubule nucleation and spindle-like structures in M-phase extract. We propose that RCC1 generates a high local concentration of Ran-GTP around chromatin which in turn induces the local nucleation of microtubules.

Two E2F sites control growth-regulated and cell cycle-regulated transcription of the Htf9-a/RanBP1 gene through functionally distinct mechanisms.

The gene encoding Ran-binding protein 1 (RanBP1) is transcribed in a cell cycle-dependent manner. The RanBP1 promoter contains two binding sites for E2F factors, named E2F-c, located proximal to the transcription start, and E2F-b, falling in a more distal promoter region. We have now induced site-directed mutagenesis in both sites. We have found that the distal E2F-b site, together with a neighboring Sp1 element, actively controls up-regulation of transcription in S phase. The proximal E2F-c site plays no apparent role in cycling cells yet is required for transcriptional repression upon growth arrest. Protein binding studies suggest that each E2F site mediates specific interactions with individual E2F family members. In addition, transient expression assays with mutagenized promoter constructs indicate that the functional role of each site is also dependent on its position relative to other regulatory elements in the promoter context. Thus, the two E2F sites play opposite genetic functions and control RanBP1 transcription through distinct molecular mechanisms.

Interactions with single-stranded and double-stranded DNA-binding factors and alternative promoter conformation upon transcriptional activation of the Htf9-a/RanBP1 and Htf9-c genes.

The murine Htf9-a/RanBP1 and Htf9-c genes are divergently transcribed from a shared TATA-less promoter. Transcription of both genes is initiated on complementary DNA strands and is controlled by cell cycle-dependent mechanisms. The bidirectional promoter harbors a genomic footprint flanking the major transcription start site of both genes. Transient promoter assays showed that the footprinted element is important for transcription of both genes. Protein-binding experiments and antibody assays indicated that members of the retinoid X receptor family interact with the double-stranded site. In addition, distinct factors interact with single DNA strands of the element. Double-stranded binding factors were highly expressed in liver cells, in which neither gene is transcribed, while single-stranded binding proteins were abundant in cycling cells, in which transcription of both genes is efficient. In vivo S1 analysis of the promoter depicted an S1-sensitive organization in cells in which transcription of both genes is active; S1 sensitivity was not detected in conditions of transcriptional repression. Thus, the same element is a target for either retinoid X receptor factors, or for single-stranded binding proteins, and form distinct complexes in different cellular conditions depending on the DNA conformation in the binding site.

Deregulated expression of the RanBP1 gene alters cell cycle progression in murine fibroblasts.

RanBP1 is a molecular partner of the Ran GTPase, which is implicated in the control of several processes, including DNA replication, mitotic entry and exit, cell cycle progression, nuclear structure, protein import and RNA export. While most genes encoding Ran-interacting partners are constitutively active, transcription of the RanBP1 mRNA is repressed in non proliferating cells, is activated at the G1/S transition in cycling cells and peaks during S phase. We report here that forced expression of the RanBP1 gene disrupts the orderly execution of the cell division cycle at several stages, causing inhibition of DNA replication, defective mitotic exit and failure of chromatin decondensation during the telophase-to-interphase transition in cells that achieve nuclear duplication and chromosome segregation. These results suggest that deregulated RanBP1 activity interferes with the Ran GTPase cycle and prevents the functioning of the Ran signalling system during the cell cycle.

Expression of the murine RanBP1 and Htf9-c genes is regulated from a shared bidirectional promoter during cell cycle progression.

The murine Htf9-a/RanBP1 and Htf9-c genes are divergently transcribed from a bidirectional promoter. The Htf9-a gene encodes the RanBP1 protein, a major partner of the Ran GTPase. The divergently transcribed Htf9-c gene encodes a protein sharing similarity with yeast and bacterial nucleic acid-modifying enzymes. We report here that both mRNA species produced by the Htf9-associated genes are regulated during the cell cycle progression, peak in S phase and decrease during mitosis. Transient expression experiments with reporter constructs showed that cell cycle expression is controlled at the transcriptional level, because the bidirectional Htf9 promoter is down-regulated in growth-arrested cells, is activated at the G1/S transition and reaches maximal activity in S phase, though with a different efficiency for each orientation. We have delimited specific promoter regions controlling S phase activity in one or both orientations: identified elements contain recognition sites for members belonging to both the E2F and Sp1 families of transcription factors. Together, the results suggest that the sharing of the regulatory region supports co-regulation of the Htf9-a/RanBP1 and Htf9-c genes in a common window of the cell cycle.